PROJECT SUMMARY DNA double-stranded breaks (DSBs) are the most toxic DNA lesions, and in mammalian cells they are predominantly repaired via the non-homologous end-joining (NHEJ) pathway. Upon formation of a DSB, p-53 binding protein-1 (53BP1), a chromatin modulator, forms higher order structures (foci) and which act as platforms to recruit DSB repair proteins to damaged chromatin and promote NHEJ by blocking end resection. Several human syndromes including severe combined immunodeficiency, and an increased sensitivity to ionizing radiation (IR) and chemotherapeutic drugs result from mutations in NHEJ proteins. Additionally, deficiencies in NHEJ may also result in mutagenic alternative end-joining (a-EJ) DSB repair, which is established in some forms of cancer. Despite much progress in the field, the organization and kinetics of NHEJ and a-EJ factors remain undefined inside the cell. Furthermore, fundamental mechanisms of repair foci and their effects on NHEJ remain poorly understood. These gaps are due to inherent limitations of conventional ensemble methods. Here, I propose to resolve these knowledge gaps by defining the molecular mechanism of the human NHEJ repair process at the level of individual 53BP1 foci via single molecule techniques such as single-molecule tracking (SMT) and stochastic optical reconstruction microscopy (STORM). These approaches will provide novel information that may be applicable to future therapy development of PARP inhibitors and NHEJ-related diseases such as XLF deficiency syndrome. The aims of the proposal are to (1) establish the kinetics and organization of NHEJ factors within individual 53BP1 foci, and (2) determine the role of 53BP1 foci in regulating the kinetics and organization of the DSB repair machinery.